Abstract

The project aimed at designing self-assembling magnetic iron particles under a magnetic field, including maghemite (Fe2O3) and magnetite (Fe3O4), for small interference RNA (siRNA) delivery then provided cell protection to neurons. Previous work has shown that magnetic iron nanoparticles are effective carriers for gene delivery in cells. We explored the possibility of using magnetic iron nanoparticles designed for delivery of N-methyl-D-aspartate receptor 2B(NR2B)-specific small interference RNA (siRNA) into neuronal cells, in which the effects has been previously proved to enhance cell survival and ameliorate the symptoms in Parkinsonian models [1]. Previous studies demonstrated that the magnetic field could facilitate the particle internalization through endocytosis so as to increase the effectiveness of siRNA uptake [2]. In the present study, magnetic nanoparticles was allowed to self assemble under a magnetic field, and siRNA was conjugated with by electrostatic force. The efficiency in terms of signal intensity was examined with a laser scan confocal microscope. Our findings supports the self-assembling magnetic nanoparticles are useful for the delivery of NR2B siRNA, which could achieve the goal of neuroprotection by regulating the NR2B protein expression. They are effective and efficient. The present findings also demonstrate a high potential in future applications of drug delivery.

Backgrounds

In recent years, since nanoparticles provide a suitable means of localized or targeted gene as well as drug delivery, more and more efforts have been devoted to nanotechnology that allows for manufacture, manipulation and control of the structure in the nanometer size range. New materials emerging from the field of nanotechnology take on novel properties and functionalities to offer targeted delivery of biomolecules, to improve bioavailability, to sustain drugs or gene effects in specific site, to prevent degradation of therapeutic agents by enzymatic activities (proteases and nucleases), especially of protein, peptide, and nucleic acids drugs [3].

The investigation of magnetic nanoparticles for target transduction of gene and drugs began 30 years ago. Major breakthroughs have been made and innovative ideas bloom in particle design as well as synthesis, however, few in vitro experiments to evaluate efficacy of magnetic iron nanoparticle-based drug and gene delivery have taken place, especially in electrically excitable neuronal cells [2]. Since biocompatibility and cytotoxicity of nanometer-sized material varies among different cell types and magnetic targeting is not likely to be effective in all situations, only with further development and verification it should provide useful tool for the effectiveness treatment of a variety of disease.

Previous work has shown that magnetic iron nanoparticles are effective carriers for gene delivery in cells [2]. We explored the possibility of using magnetic iron nanoparticles designed specifically for delivery of N-methyl-D-aspartate receptor 2B(NR2B)-specific small interference RNA (siRNA) into neuronal cells. The dopamine beta hydroxylase active, acetylcholinergic, glutamatergic neuroblastoma SH-SY5Y cell line is utilized as model neurons for us to test the siRNA-mediated gene knockdown. Glutamate is one of the key players of excitatory neurotransmitter in signal transductions between neuronal cells, the overactivation of which through direct or indirect pathways, such as glutamatergic transmission, is thought to contribute to the development of some motor impairment of Parkinson’s disease (PD)[4]. N-methyl-D-aspartate receptor 2B(NR2B) subunit is found to be the dominant NR2 subunit in ionotropic glutamate receptors most commonly expressing in the subpopulations of striatal neurons [5,6]. The administration of NR2B-specific siRNA leads to a significant reduction in NR2B protein levels [1]. Previous studies have proved the neuroprotective effects of NR2B-specific siRNA in effective amelioration of behavioral symptoms in Parkinsonian models, which is with high potential to be developed into a new therapy for PD remedy.

Project Overview

We conduct in vitro experiments to develop a simple yet effective method for self-assembling magnetic nanoparticle-based gene delivery. Our mechanism is to conjugate siRNA with magnetic iron nanoparticles by electrostatic interactions between particle surface and the therapeutic agents, and allow self-assembly of nanoparticles under magnetic field generated by high-field, high-gradient conventional magnets. The pattern formation of nanoparticles can be captured by upright microscope. Followed by immunofluorescent treatment, the gene silencing effects in terms of signal intensity are detectable by direct imaging with Confocal Laser Scanning Microscope (CLSM). We have focused on NR2B-specific siRNA delivery into neuronal cells and visualized the efficiency of magnetic iron particles in different concentrations by analyzing high-resolution images obtained from CLSM.

We propose the using of magnetic field generated by conventional magnet is able to induce the self-assembly of iron magnetic particles to form special unipolar stacking pattern, which could enhance the localization of siRNA, direct the gene delivery, facilitate internalization of nanoparticles through endocytosis into cells, and hence, improve the overall targeting efficiency of siRNA without reducing their therapeutic efficacy. We believe the simplicity and feasibility of our approach provides a promising approach for future drug delivery application.

Project Highlights

Feasibility and low cost

As conventional magnet is easy to be found in market with a relatively low price, and Fe3O4/γ-Fe2O3 nanoparticles are easy to be synthesized in laboratory all over the world, the effort-saving, money-saving, yet effective approach demonstrates a high potential in future application of drug delivery in therapeutic use.

Real in vitro experiment being conducted

With theoretical support for our ideas, we conduct in vitro experiment to evaluate the cytotoxicity of our nanoparticles and prove the efficiency of self-assembled iron nanoparticles under magnetic field in siRNA delivery.

Promising future application

Using neuroprotective NR2B-specific siRNA in SH-SY5Y neuronal cell by magnetic nanoparticle based transfection for efficient silencing of NR2B gene is proved to be effective, which provides a therapeutically significant means for amelioration of behavioral symptoms in Parkinsonian models as well as ischemic stroke, Huntington’s disease, etc. Our study proposes potential application of stacked iron nanoparticles under magnetic field for delivery of therapeutic agents to the cells and tissue efficiently without reducing their therapeutic efficacy.

Design

Self-assembly and characterization of iron nanoparticles

We use simple, basic inorganic iron chemistry to synthesize our Fe3O4/γ-Fe2O3 nanoparticles according to Massart’s method [7]. The co-precipitation of ferrous and ferric ion solutions enables the formation of monosized iron nanoparticles. Maghemite (Fe2O3) and magnetite (Fe3O4), are least likely to cause any health hazard since iron ions widely exist in human body, so any leakage of metal should not induce significant side effects. As materials in biomedical application are required to be biocompatible, of which the Fe3O4、γ-Fe2O3 with low cytotoxicity are rational candidates.

We apply magnetic field generated by high-field, high-gradient conventional magnets, which are easy to be purchased in market and handled in laboratory, to enable self-assembly of our magnetic iron nanoparticles, and the unipolar orientation and numerous stacking pattern formed in different concentration are observed with upright light microscope, images are captured. We propose that the use of iron nanoparticles and special pattern of magnetic iron nanoparticles induced by conventional magnetics are able to enhance overall efficiency of gene-delivery and demonstrate the significance with in vitro experiments.

Magnetic nanoparticle-mediated gene knockdown

Small interfering RNA, also known as silencing RNA is a class of double- stranded RNA with 20-25 nucleotides in length, which is commonly utilized as molecule tool to trigger target gene knockdown by RNA interfering (RNAi) pathway. siRNA interferes with specific gene expression by complementary binding to messenger RNA(mRNA), degrading mRNA, thus block successfully expression of genes.

In our experiment, NR2B-specific siRNA is conjugated with magnetic iron nanoparticles through electrostatic interaction between particle surfaces and negatively charged siRNA, through which, siRNA is under protection of nanoparticles from degradative effects of nuclease and other enzymatic activities. The conjugation may result in improved siRNA efficiency by better internalization across cell membrane, reduced intracellular degradation of siRNA-nanoparticle complexes by nucleases, increase in vivo stability and reduce their immunogenicity. Furthermore, the magnetic field generated by high-field, high-gradient conventional magnets could facilitate the particle internalization through endocytosis so as to increase the effectiveness of siRNA uptake.

Immunofluorescence analysis

Immunofluorescence technique is utilized in our experiments to detect the presence NR2B protein by exploiting the principle of specific binding of antibodies to antigen in biological tissues. The gene expression level of NR2B is visualized by analyzing high-resolution images obtained from CLSM and data analysis is conducted correspondingly.